Characterizing the extent human milk folate is buffered against maternal malnutrition and infection in drought‐stricken northern Kenya

Abstract Objectives Folate is an essential nutrient fundamental to human growth and development. Human milk maintains high folate content across the maternal folate status range, suggesting buffering of milk folate with prioritized delivery to milk at the expense of maternal depletion. We investigated whether and how the extent of this buffering may diminish under prolonged nutritional and/or disease stress, while taking into consideration infants' varying vulnerability to malnutrition‐related morbidity/mortality. Methods A cross‐sectional study analyzed milk specimens from northern Kenyan mothers (n = 203), surveyed during a historic drought and ensuing food shortage. Multiple regression models for folate receptor‐α (FOLR1) in milk were constructed. Predictors included maternal underweight (BMI < 18.5), iron‐deficiency anemia (hemoglobin <12 g/dl and dried‐blood‐spot transferrin receptor >5 mg/L), folate deficiency (hyperhomocysteinemia, homocysteine >12 or 14 μmol/L), inflammation (serum C‐reactive protein >5 mg/L), infant age and sex, and mother‐infant interactions. Results In adjusted models, milk FOLR1 was unassociated with maternal underweight, iron‐deficiency anemia and inflammation. FOLR1 was positively associated with maternal folate deficiency, and inversely associated with infant age. There was interaction between infant age and maternal underweight, and between infant sex and maternal folate deficiency, predicting complex changes in FOLR1. Conclusions Our results suggest that mothers buffer milk folate against their own nutritional stress even during a prolonged drought; however, the extent of this buffering may vary with infant age, and, among folate‐deficient mothers, with infant sex. Future research is needed to better understand this variability in maternal buffering of milk folate and how it relates to folate status in nursing infants.


| INTRODUCTION
Mothers' milk nourishes and protects infants by delivering essential nutrients, immune factors, and other bioactive compounds (Miller et al., 2013). The macronutrient and energy content in human milk, and more generally the milk of primates, are buffered against shortterm maternal nutritional stress (Miller et al., 2013;Prentice et al., 1981;1994;Villalpando & del Prado, 1999). This buffering of milk nutrients is possible through a combination strategy to draw nutrients from both dietary sources ('income') and somatic nutrient reserves ('capital') for milk synthesis (Hinde et al., 2009). This strategy is not shared by other mammals, such as rodents, whose milk nutrients originate (Black, 2008) predominantly in their 'income' (Hinde et al., 2009).
Here, we expand on the maternal buffering hypothesis by evaluating whether human milk micronutrient content (folate) may be similarly buffered against maternal nutritional or disease stress. Using data from a harsh environment with prolonged drought, we characterize the pattern of variation in milk folate content to allow for an understanding of the extent of maternal buffering of milk folate against different forms of maternal nutritional and infectious disease stress. We integrate the maternal buffering with a complementary concept-the 'protective' hypothesis-that milk nutrient content increases in proportion to infant needs (e.g., needs for protection against malnutrition or infectious disease [Breakey et al., 2015;Fujita et al., 2019;Fujita, Paredes Ruvalcaba, Wander, et al., 2018]). To this end, we evaluate whether and how maternal and infant characteristics may interact to affect milk folate content.

| Maternal buffering
The maternal buffering hypothesis asserts that evolution has shaped primate and human lactation to be capable of buffering milk nutritional value against maternal malnutrition (Miller et al., 2013;Prentice et al., 1981;1994;Villalpando & del Prado, 1999). Buffering is possible through mobilization of maternal body reserves, which, while they last, can continue to nourish milk when dietary sources of nutrients dwindle (Hinde et al., 2009). Previous studies have provided some support for maternal buffering of milk energy content (Fujita, Paredes Ruvalcaba, Wander, et al., 2018;Lönnerdal, 1986;Mandel et al., 2005). Less is known about maternal buffering of milk micronutrient content.
Micronutrients in milk have been categorized into two groups based on the degree to which their concentrations vary in relation to maternal status and intake. Group I nutrients are those that tend to decrease when maternal status or intake decreases, and include vitamin B6, vitamin B12, and retinol, among others (Allen, 2012). Group II nutrients are more robust to maternal malnutrition or inadequate dietary intake. These include folate (Allen, 2012), which appears to maintain remarkably high concentrations. Researchers have interpreted these findings to mean that maternal physiology gives high priority to delivering folate to infants, even at the expense of maternal folate depletion (Allen, 2012;Metz, 1970;Salmenpera et al., 1986). Existing literature therefore characterizes milk folate as fairly invariant.
For anthropologists interested in human adaptability, this characterization of milk folate as invariant is a hypothesis worthy of additional scrutiny. Although milk folate content may be generally robust to the effects of maternal folate deficiency among the populations well-represented in the literature, less is known about whether or how milk folate may vary across the full range of human variation, including contexts of severe and multifaceted ecological stress that can result in prolonged periods of poor nutrition among mothers.
Although a stable supply of milk folate in such settings is important for infants, mothers also need folate for their immediate physiological needs, long-term health, and future reproduction (Bailey & Gregory, 1999;Bartley et al., 2005;Jablonski & Chaplin, 2000;Tamura & Picciano, 2006). The maternal costs of buffering milk folate (i.e. less folate available for mothers themselves) are likely to be high and influenced by the extent and duration of maternal malnutrition.
This leads us to expect that the extent of maternal buffering of milk folate should vary with maternal folate nutrition and other stressors.

| Maternal protection
The protective framework views milk content as maternal effort to protect the infant against nutrient shortfalls and/or infectious diseases, and therefore, expects maternal delivery of milk content to vary with differing infant needs (Breakey et al., 2015;Fujita et al., 2019).
From this perspective, we expect mothers to increase delivery of milk nutrients or protective factors as infants' risk for mortality from malnutrition and/or infectious diseases increases (and conversely to decrease delivery as risk decreases).

| Folate nutrition
Folate is an essential nutrient, utilized by nearly all organisms for multiple biological functions, including nucleic acid and protein biosynthesis (Gorelova et al., 2019). Folate is important for fetal and infant development and sustained growth (e.g., via methylation of DNA and histones on the genes that code for development [Tamura & Picciano, 2006]). Folate is also important for competent cell-mediated immune responses (Courtemanche et al., 2004).
Given the fundamental biological function of folate, it is not surprising that fetal and infant demand for folate are elevated. Folate needs are correspondingly high for mothers during pregnancy and lactation (Jablonski & Chaplin, 2000). Folate deficiency among women at pre-conception increases the risk of congenital defects and pregnancy failure (Bartley et al., 2005). For this reason, a synthetic form of folate, folic acid, is widely utilized for maternal supplementation during pregnancy to prevent neural tube defects in offspring (Tamura & Picciano, 2006).
Postpartum, breastfed infants obtain folate through milk. Compared to other dietary sources of folate (e.g. legumes, cereals, green vegetables), milk folate has superior bioavailability. Previous research has found that milk folate concentrations are maintained even among mothers with folate deficiency, and are unaffected by folic acid supplementation (Cooperman et al., 1982;Houghton et al., 2009;Mackey & Picciano, 1999;Trugo et al., 1988). Maintaining an adequate milk folate concentration to support infant development may come at the expense of maternal well-being (O'Connor et al., 1997;Tamura & Picciano, 2006). However, low milk folate concentrations can occur when maternal folate levels are abnormally low (O'Connor et al., 1997) or in cases of experimentally induced folate deficiency (Metz, 1970).
The milk folate-FOLR1 complex is a highly efficient means of folate transfer to infants that sets mothers' milk apart from other folate sources. In the mammary gland, FOLR1 proteins function to concentrate folate multifold from maternal plasma to milk (FOLR1 can be 10-fold to 1000-fold more concentrated in milk than blood plasma R&D, 2011;Tamura et al., 2009]; the magnitude of difference likely depends on the method of analysis; see [Nygren-Babol & Jagerstad, 2012]). FOLR1 stabilizes folate, prevents enzymatic degradation, and increase folate bioavailability to infants (Ford, 1974;Mason & Selhub, 1988;Nygren-Babol & Jagerstad, 2012).
FOLR1 also prevents bacteria in the infant gut from accessing maternal folate, similar to lactoferrin sequestering iron from microorganisms (Trugo et al., 1988). In milk, folate and folate binding protein are highly positively correlated (r = 0.71) among US mothers (Selhub et al., 1984;Tamura et al., 2009). FOLR1 preserves well under cryogenic temperatures and tolerates multiple freeze-thaw cycles (Balion, 2011); the stability of FOLR1 makes it an excellent biomarker for milk folate content.

| Milk folate content variation by maternal nutrition and inflammation
Consensus is currently lacking regarding the effects of different aspects of maternal nutrition (other than folate status), inflammation and infection on milk folate concentration. There is some evidence that maternal delivery of milk folate may be influenced by non-folate maternal nutrition (Donangelo et al., 1989;Oconnor et al., 1987). A study in Brazil found milk folate content was positively associated with maternal serum vitamin B12, zinc, and albumin even though it was unassociated with maternal serum folate, iron, and ferritin (Donangelo et al., 1989). A study in Mexico found no difference in milk folate concentrations by maternal iron deficiency or low blood folate (Khambalia et al., 2006;Villalpando et al., 2003), contradicting prior research with animal models suggesting that milk folate secretion can decline during iron deficiency (Oconnor et al., 1987).
While we are aware of no studies that specifically investigated the effect of maternal inflammation or infection on milk folate content, the broader literature provides clues that inflammation or infection may influence milk folate. This includes the reported associations between some chronic inflammatory bowel conditions and low blood folate, likely due to low nutrient absorption (Stabler, 2010), and a positive association between serum C-reactive protein (CRP, an acute phase reactant and biomarker of inflammation) and folate deficiency among lactating indigenous women in Panama (Gonzalez et al., 2017).
Furthermore, our previous studies have linked elevated CRP among lactating mothers to altered nutrient content in their milk, including fat (Fujita, Paredes Ruvalcaba, Wander, et al., 2018) and retinol  in Kenya, although observations in Malawi contradict the latter (Dancheck et al., 2005).
Lack of consensus also extends to the effect of infant characteristics on milk folate content. Studies report increases, decreases, or no change in milk folate contents in association with infant age (e.g., [Donangelo et al., 1989;Mackey & Picciano, 1999;Tamura et al., 2009]), with discrepancies likely due in part to differences in maternal folate status (Tamura et al., 2009). Clarifying the roles maternal nutritional and disease status play in milk folate content can help us better understand variation in postnatal folate transfer in ecological context, with important implications for infant growth and development.

| Maternal buffering and protection combined: hypotheses
We expect milk folate delivery to be buffered against maternal nutritional or disease stress, but the extent of buffering to vary in ways F I G U R E 1 Hypothesized milk folate content variation patterns from the combined perspective of maternal buffering and protection reflecting maternal protective effort. Buffering milk folate entails high opportunity cost to undernourished mothers (i.e. less folate available to support their own health), and therefore we broadly predict that mothers with nutritional deficiencies or inflammation/infection will imperfectly buffer milk folate. We further broadly predict more protective effort (i.e. higher milk folate) for infants who are more vulnerable to physiological effects of folate shortfalls and have higher needs for maternal protection (e.g., younger infants undergoing rapid cellular processes requiring folate [Black, 2008]). Finally, we predict the difference by infant vulnerability to be more apparent among mothers with nutritional deficiencies or inflammation/infection (due to more robust buffering in effort to protect more vulnerable infants and attenuated buffering for less vulnerable infants) than mothers under less stress;

| Study objectives
We conducted a secondary analysis of the cross-sectional data from 203 predominantly normal-to-underweight, seemingly healthy breastfeeding mothers within 20 months postpartum, collected during drought-induced food scarcity in northern Kenya. The study objectives were to evaluate whether milk folate delivery is buffered against maternal nutritional or disease stress; whether milk folate buffering is incomplete among some mothers; and whether milk folate delivery exhibits maternal protection (enhanced delivery to infants most vulnerable to ill effects of folate deficiency).

| MATERIALS AND METHODS
The study assessed associations of milk folate with maternal nutritional status and elevated inflammation (a likely indicator of infectious disease), infant age and sex, and interactions between maternal and infant variables.
The original data collection occurred during the 2006 Horn-of-Africa drought, one of the most serious droughts during the recent history of East Africa. The drought caused major loss of livestock and agricultural crops in northern Kenya. This intensified food insecurity and many residents were dependent on drought relief foods (Fujita, 2008). In addition to two consecutive years of failed rainfall (2004)(2005), the drought was compounded by conditions leading into it, including poor livestock health, decimated herd sizes, and high food prices (Marsabit District, 2006;USAID, 2018;World Health Organization, 2006). The government response (famine food relief) was also slow, contributing to the severity of the problem (World Health Organization, 2006). As such, food insecurity had been chronically high for at least 2 years at the time of data collection. The surveyed mothers were 1-20 months postpartum, meaning all carried their pregnancies during the extended period of failed rainfall and food scarcity. The original study did not collect any data on gestation or birth weight. In the present study therefore we assume all pregnancies were similarly affected by the drought and famine. In reality, however, the impact of food and water scarcity likely differed between mothers, depending on the timing of pregnancy relative to the timing of socioecological events such as the onset of famine relief distributions (USAID, 2018;World Health Organization, 2006). Data from the original and subsequent studies have revealed high rates of maternal malnutrition, anemia, and inflammation/infection (Fujita et al., 2011;2012;Fujita & Wander, 2017;Paredes Ruvalcaba et al., 2020). For the present study, we drew on a convenience subsample of participants for whom all relevant data were complete, of the random sample of 241 mothers representing three communities in the original study. The study was approved and overseen by the institutional review boards of the University of Washington and Kenya Medical Research Institute. All mothers provided informed consent. The analysis of de-identified milk specimens and data presented here required no further approvals.

| Milk FOLR1 concentration estimation
The milk utilized for the determination of milk FOLR1 were foremilk specimens expressed manually by mothers into a disposable cup (assisted by female staff, as needed). Mothers were asked to fast overnight and refrain from use of one breast for overnight feeding, for the original purpose of determining milk retinol concentration, as described elsewhere (Fujita, 2008;Fujita et al., 2011). Upon collection, the milk was immediately transferred to an opaque bottle to minimize UV exposure and frozen in liquid nitrogen. This sampling procedure (morning specimens of fasting milk from the breast not nursed overnight) allowed reducing the influence of immediate meals, breastfeeding, and possible diurnal patterns in milk synthesis on milk retinol in our original research, and on milk FOLR1 in the present study.
The specimens were maintained frozen at cryogenic temperatures, except for undergoing two freeze-thaw cycles. The aqueous fraction of milk specimens was obtained by triple-centrifuging thawed and homogenized milk at 4 C, as described elsewhere . Milk FOLR1 concentrations were estimated in 2017 in the Biomarker Laboratory for Anthropological Research at Michigan State University with an enzyme immunoassay kit validated for use in human milk (Quantikine ELISA Human FOLR1 Immunoassay DFLR10 [R&D, 2011]). The intra assay CVs were ≤7.1% and the inter-assay CVs were ≤6.8% for the controls of low and high concentrations across seven plates.

| Infant age and sex
Infant age (in months) was determined from the infant's date of birth reported in a one-on-one interview with the mother. Infant sex (female/male) was also based on maternal report. Infant vulnerability to malnutrition and infectious disease was considered higher in infants of younger ages and male sex. We consider vulnerability here specifically with regard to physiological needs for folate; younger infants undergo more rapid cell divisions for growth and development than older infants (Black, 2008). As such, shortfalls in Male infants tend to be more susceptible to infection and growth faltering under stress (Naeye et al., 1971;Stinson, 1985) than female infants of the same age.

| Statistical analysis
We fit a series of multiple regression models using natural logtransformed milk FOLR1 as the dependent variable. Predictors included maternal underweight, iron deficiency anemia, hyperhomocysteinemia, inflammation, infant sex, and infant age (centered at mean). Models tested the interactions between maternal variables and infant age/sex. When an interaction was apparent, we conducted a joint F-test to evaluate the joint significance of the main-effect and interaction terms.
Dichotomous parameterizations of biomarker variables (e.g., iron deficiency anemia) represent conditions with meaningful biochemical or physiological consequences for human health. Continuous predictor infant age was centered to facilitate interpretation of the coefficients of other predictors, i.e. as their effects on FOLR1 at the mean infant age.
Control variables, which might be associated with both milk FOLR1 and maternal nutrition (Corbitt et al., 2019;Fujita, 2008;Fujita et al., 2019) included: maternal age and parity, postpartum resumption of menstruation (yes/no), breastfeeding frequency, complementary feeding status (yes/no), milk total protein concentration (measured by microBCA assay [Corbitt et al., 2019]), socioeconomic status (a dichotomous variable for low status by a combination of selfreported poverty and below-median household-owned land size/ livestock holding), and community. Since FOLR1 is a specific type of protein out of numerous proteins in milk (and highly correlated with total milk protein), we adjusted models for milk total protein to interpret the coefficients of the predictors as their effects on FOLR1 specifically, rather than their general associations with milk proteins.
These variables were entered into regression models one at a time and retained if they either substantially altered one or more predictors' individual coefficients or improved model fit (evaluated with the Bayesian information criterion, BIC). If the entered variable did neither, then it was not retained in the model.
Resulting models were assessed for multicolinearity using variance inflation factor and other violations of regression assumptions using residual plots and Cook's distance for outlier influence/leverage. We report raw and standardized regression coefficients (β) from the models, and use a probability less than 0.10 as the evidence for association, favoring the power of type II error (Kim & Choi, 2021)

| Milk FOLR1 by infant characteristics
In the main effect models, infant age was inversely associated with milk FOLR1 (p < 0.001 in both models 1a, 1b), predicting a 2% T A B L E 2 Regression models for milk folate receptor-α (FOLR1, log-transformed) by maternal nutritional/inflammation status and infant characteristics using lower (A) and higher (B) hyperhomocysteinemia cutoffs

| Milk FOLR1 by mother-infant interaction
Of the eight possible interactions, two were noteworthy (Table 2 interaction models 2 and 3; all other interaction models are shown in Supplemental Information, Tables S1 and S2). First, there was an interaction (p < 0.001) between infant age and maternal underweight (Joint F test F (2,194)  ished. The pattern of this interaction is illustrated in Figure 3. Milk folate delivery was higher for sons than daughters (the difference of 11%) among mothers with hyperhomocysteinemia, while among mothers without hyperhomocysteinemia, milk FOLR1 delivery was higher for daughters than sons (the difference of 7%).

| DISCUSSION
We used data from northern Kenya to investigate whether maternal delivery of folate to milk may be buffered against maternal nutritional and infectious disease stress and proactively calibrated to protect infants against similar stresses. We tested a set of hypotheses combining the ideas of maternal buffering and maternal protective effort.
We expected milk folate to be generally buffered against maternal nutritional or disease stress, but we further expected that the extent of buffering would vary in ways reflecting the balance between cost and benefit of buffering ( Figure 1). Namely, we generally predicted that mothers raising younger or male infants would deliver more folate to milk, particularly among mothers with nutritional deficiencies or inflammation/infection, who face the highest costs to buffering milk folate. Our results did not fully conform to the pattern of variation we expected; however, we did observe patterns that are indicative of maternal buffering and protective efforts, particularly for infants with elevated needs (i.e. younger/male). The interactive effects we observed may suggest a more complex relationship between maternal condition and infant vulnerability than we predicted.

| Buffered milk folate (mothers maintain, or increase, folate delivery to milk)
Our findings generally support the maternal buffering hypothesisusing FOLR1 as a measure of folate, milk folate was unaffected by maternal underweight, iron deficiency anemia, or inflammation, and was positively associated with hyperhomocysteinemia (likely representing folate deficiency). The lack of evidence for compromised milk folate delivery among mothers with underweight, iron deficiency anemia, infection, or hyperhomocysteinemia suggests that mothers of northern Kenya during a serious drought and resulting food scarcity generally buffered milk folate against their own nutritional and disease stress-mothers could maintain their folate delivery to milk regardless of malnutrition or inflammation/infection. This extends the evidence for maternal buffering of human milk beyond energy content (Fujita, Paredes Ruvalcaba, Wander, et al., 2018;Lönnerdal, 1986;Mandel et al., 2005). This also corroborates nutritional scientists'  (Hill & Kaplan, 1999;Stearns, 1992) among underweight mothers. We should note that predicted milk FOLR1 values for underweight mothers in the early postpartum period are likely to be based on few cases (as pregnancy weight gain makes it unlikely that even an undernourished mother would fall under BMI of 18.5, the definition for underweight, in the early postpartum weeks), and so differences in FOLR1 among mothers at young infant ages should be interpreted with caution. However, distinct patterns became apparent at later infant ages: among non-underweight (better nourished) mothers milk folate decreased with infant age.
We also found some evidence that milk folate might have been considerably higher for mothers with hyperhomocysteinemia who were raising sons, compared to daughters. This may represent protection for male infants, who may be, relative to female infants, more susceptible to infectious diseases (Naeye et al., 1971) and sensitive to environmental stress during growth (Stinson, 1985). In the context of drought and heightened nutritional and disease stress in northern Kenya, these protective benefits may outweigh the cost to the mother (e.g., folate deficiency). Our analyses were also limited by the number of hyperhomocysteinemia cases by the >14 μmol/L homocysteine cut-point (n = 16).

| When mothers may increase milk folate delivery/buffering: cost-benefit analysis
We may have missed real but subtle patterns in these models.
Another possible limitation relates to the long-term storage of milk specimens, which may have affected FOLR1 concentrations. This is, however, unlikely-a study indicated that the analytic recovery of folate in blood serum stored for 29 years at À25 C was approximately 80% (Hannisdal et al., 2010). FOLR1 is more robust than folate, and the present project utilized specimens stored for a shorter duration (11 years) at substantially lower temperatures <À70 C, more favorable for minimizing the long-term storage effect on biological specimens. FOLR1 also tolerates multiple (at least three) freeze-thaw cycles when frozen cryogenically (Balion, 2011).
In interpreting the results, the practical importance of the effect size should ideally be appraised against what may be a biologically meaningful effect size (Wasserstein et al., 2019). The paucity of information on human milk FOLR1 (because few previous studies quantified FOLR1 specifically) presents a challenge in this regard. However, the broader literature on blood FOLR1 concentrations provides a general benchmark to gauge the possible biological meaning of differences in milk FOLR1 we report in this study. A 22% difference in blood serum FOLR1 concentration has been attributed to active periodontitis vs. healthy control (Alkan et al., 2019), while a difference of 34% has been observed between mothers who did (vs. did not) experience a neural tube defect birth (Celik et al., 2014). Gauged against these values, the magnitude of effects we reported here (e.g., 7%-15%) may have important implications for infants' health, growth and development long term.
Finally, we have used FOLR1 as a biomarker for milk folate, rather than measuring milk folate itself. This is justified because milk folate is less robust to storage; however, it does introduce opportunity for error if the association between milk folate and FOLR1 does not hold under some conditions.

| Future directions
Studies on buffering of milk nutrient content, including folate, are needed to understand whether and under which contexts buffering occurs and to what extent. Such studies need to investigate communities subject to varying degrees of nutritional stress, including severe stresses such as those of a prolonged drought. Furthermore, future study is needed to understand whether the magnitude of the effects we report here are biologically meaningful, and how maternal buffering relates to folate status in nursing infants. Investigation of interactions in the mother-infant dyad are a necessary step toward better understanding of the impact of context on nutrition interventions (Raiten et al., 2021).

| CONCLUSIONS
Our findings support the maternal buffering hypothesis, and reveal complex interactions between maternal and infant factors in milk folate content. Mothers generally seem to buffer milk folate content against their own nutritional or disease stress. Milk folate does vary with infant characteristics (age and sex) in ways arguably congruent with the protective hypothesis. Further research is needed to clarify the diachronic dimension in milk folate variation, such as whether elevated maternal delivery of milk folate may be triggered by, or contribute to, maternal folate deficiency, and to determine the impact of milk folate on infant folate nutrition and health, growth, and development outcomes.